To enhance the impact of

To enhance the impact of GD2 as an immune target in this cancer, we investigated a novel strategy to upregulate expression on the cell surface of EwS cells by an epigenetic agent, based on the following rationale. Biosynthesis of GD2 and other gangliosides during organ development is driven by stage-specific transcriptional activation of glycosyl transferases and underlies epigenetic regulation. Epigenetic reprogramming is highly relevant also in the pathology of EwS.24, 25, 26, 27, 28, 29 An important epigenetic regulator in EwS is Enhancer of Zeste Homolog 2 (EZH2), the catalytic component of the Polycomb Repressor Complex 2 (PRC2).24, 30 EZH2 acts as a histone methyltransferase, and it silences genes involved in cell differentiation in a highly context-dependent manner, by depositing repressive histone marks at histone 3 lysine 27 (H3K27me3). High-level EZH2 expression is induced in EwS as a direct consequence of EWSR1-FLI1, and it has a central role in maintaining self-renewal and tumorigenicity.24, 30 Epigenetic plasticity by EZH2-mediated gene regulation contributes to phenotypic heterogeneity among EwS cells. We hypothesized that EZH2 is involved in the regulation of synthesis of the non-protein neuroectodermal marker GD2 in this cancer, allowing us to sensitize EwS cells to GD2-targeted cell therapy by EZH2 inhibitors.
Results
Discussion GD2 was one of the first PD-1/PD-L1 Inhibitor 3 used to retarget T cells to tumor cells by CARs,38, 39 and it remains an attractive CAR target. As an oncofetal antigen, GD2 has highly restricted normal tissue expression.11, 12 A concern has been low-level antigen expression on neuronal cells, but GD2-directed CAR T cell therapy has been safe in early clinical trials in patients with neuroblastoma, now including studies with signal-enhanced CARs, use of lymphodepleting chemotherapy, and clear evidence of activity by cytokine release and/or tumor responses (K. Straathof et al. 2018, AACR Cancer Res. abstract).16, 17, 18 Extending the therapeutic potential of GD2-specific CAR T cells beyond neuroblastoma is challenged by low levels and high heterogeneity of GD2 antigen expression in other cancers expressing GD2.9, 10, 40 Here we report a strategy by which EwS cells with low or absent GD2 expression can be sensitized to GD2-directed CAR T cell therapy. At pharmacologically relevant concentrations that effectively reduce H3K27me3, the EZH2 inhibitors GSK126 and tazemetostat significantly upregulated GD2 surface expression in a majority of GD2neg EwS cell lines, including a multilayered 3D tumor model. Following epigenetic upregulation of GD2 in this manner, EwS cells induced antigen-specific activation responses by GD2-retargeted CAR T cells, and they were effectively lysed by the CAR T cells in vitro. We did not attempt to demonstrate activity of the combination therapy in an in vivo model for the following reason: in mouse xenograft models of EwS, GD2-specific CAR T cells or CAR natural killer (NK) cells had only modest antitumor activity, despite the consistent expression of GD2.9, 10, 20 Further therapeutic elements will have to be included to break barriers in the tumor stroma against effective in vivo tumor control. CAR T cells fail to access tumors at sufficient numbers, and upon activation they upregulate immune-inhibitory molecules, such as PD-L1 and HLA-G, and they induce large populations of myeloid suppressor cells. But although reliable expression of the CAR target antigen is insufficient to establish efficacy in mouse models and in clinical settings, it remains an indispensable prerequisite for the action of CAR T cells. Expression of GD2 and its regulation by an epigenetic mechanism in EwS is well in line with the pathogenesis of this cancer. According to current models, EwS arises in MSCs as a consequence of the disease-defining chromosomal translocation. Since surface GD2 characterizes both neuroectodermal and MSCs,11, 43 expression in EwS could be a reminiscent feature of its histogenic origin. In normal MSCs, GD2 expression is associated with an immature phenotype and is lost during non-neuronal lineage differentiation,44, 45 likely by epigenetic gene repression, which has a central role in modulating ganglioside expression during normal neural development. Epigenetic gene regulation is highly relevant also in the pathogenesis of EwS. In the permissive cellular environment of the mesenchymal cell, the aberrant transcription factor EWSR1-FLI1 reprograms the epigenome to induce the oncogenic phenotype.25, 27